Modifications of solid 3-sn-phosphoglycerides

ABSTRACT

Methods for hydrolyzing solid ungranulated lysophosphatidylcholine with phospholipase A 2  are provided. Also disclosed are methods for making a lipid matrix of lysophosphatidylcholine, monoglyceride and fatty acid, and lipid matrices of particular structure.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/492,252, filed Oct. 11, 2002 and now pending, which was a nationalstage filing under 35 U.S.C. § 371 of PCT International applicationPCT/US02/32647, filed Oct. 11, 2002, which was published under PCTArticle 21(2) in English, which claims the benefit under 35 USC § 119 ofU.S. provisional application Ser. No. 60/328,660, filed Oct. 11, 2001,all of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of phospholipidhydrolysis. In particular, this invention relates to an improved methodof phospholipase A₂ hydrolysis of solid ungranulated phosphatidylcholineto produce lysophosphatidylcholine. This invention also relates to amethod of making a lipid matrix comprising lysophosphatidylcholine,monoglyceride, and fatty acid, as well as lipid matrix compositionshaving a non-lamellar structure and high viscosity.

BACKGROUND OF THE INVENTION

Enzymatic conversion of phosphatidylcholine to lysophosphatidylcholinehas been known since the early 1900's. Early investigations of thedegradation of lecithin (phosphatidylcholine) by snake venom extractsdemonstrated that the action of snake venom hemolysis is upon thelecithin portion of the cell membrane. In 1935, Hughes demonstrated thatthe hydrolysis of a unimolecular film of lecithin to lysolecithin(lysophosphatidylcholine) is dependent on factors such as pH,temperature and the surface concentration of the lecithin molecules.Packing of the lecithin molecules in the unimolecular layer greatlydecreased the rate of hydrolysis. Hanahan demonstrated that anether-soluble complex between egg phosphatidylcholine and phospholipaseA₂ resulted in the release of unsaturated fatty acid andlysophosphatidylcholine. Hydrolysis of phosphatidylcholine byphospholipase A₂ could not be detected when 95% ethyl alcohol,chloroform or petroleum ether were used as solvents. Experimentsperformed by Dawson, reported in 1963, also found that phospholipase A₂hydrolyzed phosphatidylcholine to lysophosphatidylcholine and a singlefatty acid molecule. Dawson determined that the enzymatic activity wasdependent on the presence of calcium ions, and that the addition ofether or butanol stimulated the phospholipase A₂ activity. Britishpatent 1,215,868 to Unilever Ltd. described a further modification ofthe hydrolysis of phospholipid by phospholipase A₂, conducting thereaction in the presence of fat (oils).

The processes of phosphatidylcholine hydrolysis disclosed in the priorart suffer from several shortcomings, including incomplete hydrolysisand production of unwanted side products in the hydrolysis reaction. Thedeficiencies of the prior art methods are severe because the presence ofunreacted starting materials or unwanted side products represent anunacceptable level of contaminants in the final reaction product. Theseunwanted constituents must be removed from the reaction product in orderto obtain the desired product, lysophosphatidylcholine, thusnecessitating additional purification steps.

The prior art methods described above produce a maximal yield oflysophosphatidylcholine of approximately 70% of the startingphosphatidylcholine. Dawson showed that the addition of ether wasrequired to stimulate the phospholipase A₂ activity in the hydrolysis ofphosphatidylcholine to the maximum yield of about 60-70%. The maximumyield of lysophosphatidylcholine was obtained when 8% diethyl ether(vol./vol.) in aqueous buffer was the reaction medium; using thisreaction medium a two-phase system was observed. Dawson also found that6% butanol (vol./vol.) could substitute for diethyl ether in thereaction medium to enhance yield of lysophosphatidylcholine, but ethanoland methylisobutylhexane were ineffective for increasing hydrolysis ofphosphatidylcholine. Dawson concluded that the stimulatory effect ofether (or butanol) on hydrolysis of phosphatidylcholine was probably dueto surface dilution of the closely packed phosphatidylcholine moleculesoriented at the lipid interface and a removal of inhibitory fatty acidcarbonyl groups from the interface. This conclusion was supported byevidence that addition of fatty acids inhibited the enzymatic hydrolysisof phosphatidylcholine (Dawson). Inhibition of the reaction by addedfatty acid resulted either from inhibiting the removal of the fatty acidfrom the interface, or from formation of a calcium ion—fatty acidchelate, i.e., removal of Ca²⁺ ions required for phospholipase A₂activity. Dawson believed that the removal of calcium ions was the morelikely explanation because the further addition of ether to form twophases and solubilize the additional fatty acid did not promotehydrolysis of phosphatidylcholine, whereas increasing the calciumconcentration ten fold did partially relieve the inhibition. It was alsoshown that the phospholipase A₂ enzyme purified from cobra venom wasdependent on the presence of calcium ions for hydrolysis activity. Therequirement for calcium ions in the hydrolysis reaction by phospholipaseA₂ and the association of calcium ions with fatty acids released by thehydrolysis of phosphatidylcholine is well known in the art (NovoNordisk).

Yesair described methods for the preparation of mixed lipid particlesuseful in the delivery of drugs and for providing readily absorbablecalories to an individual (U.S. Pat. Nos. 4,874,795 and 5,314,921).These methods involve the mixing of lysophosphatidylcholine,monoglyceride and fatty acid in specific molar ratios. Although easilyperformed, these previous methods use costly, isolated, highly purifiedlysophosphatidylcholine, thus adding to the expense of the final mixedlipid particle product.

Yesair subsequently described methods by which phosphatidylcholine ismore efficiently converted to lysophosphatidylcholine (U.S. Pat. No.5,716,814). The described methods result in more efficient use ofphosphatidylcholine and yield fewer unwanted side products (such asglycerophosphatidylcholine) and contaminants (such as unhydrolyzedphosphatidylcholine) in the final reaction product. The use of thedescribed methods in which the end products are in a more pure formresults in substantial cost savings and time savings due to a reducedneed for the purification of the end products. These methods require,however, the use of partially purified phosphatidylcholine in granulatedform. While ability to use of this form of phosphatidylcholinerepresents an improvement over prior methods, there remains a need toreduce the cost of production of lysophosphatidylcholine. A method whichutilizes less highly processed phosphatidylcholine as a startingmaterial would reduce the need for the use of a relatively moreexpensive granulated preparation of phosphatidylcholine as a startingmaterial, thus reducing the overall costs for the finallysophosphatidylcholine product and for mixed lipid particle productsprepared using lysophosphatidylcholine.

SUMMARY OF THE INVENTION

The invention involves improvements in enzymatic modification of3-sn-phosphoglyceride molecules, particularly hydrolysis ofphosphatidylcholine. In a preferred embodiment, the present inventionprovides methods whereby solid ungranulated blocks ofphosphatidylcholine can be converted to lysophosphatidylcholine withnearly 100% efficiency. Further, the hydrolysis of phosphatidylcholineaccording to the present invention results in production of smallquantities, if any, of unwanted side products. The present inventionprovides a method which reduces the cost of makinglysophosphatidylcholine by converting ungranulated phosphatidylcholine.

The present invention also provides lipid matrix compositions havingparticular structure and associated physical properties. Thesecompositions are useful, inter alia, as drug delivery compositions.

According to one aspect of the invention, methods for modifyingungranulated/solid matrix 3-sn-phosphoglyceride molecules are provided.The methods include forming a reaction mixture by contactingungranulated/solid matrix 3-sn-phosphoglyceride molecules with an amountof phospholipase A₂ sufficient to modify an ester bond of the3-sn-phosphoglyceride molecules, and incubating the reaction mixture tomodify the 2-acyl bond. In certain preferred embodiments, the ester bondmodification is hydrolysis. In other preferred embodiments, the3-sn-phosphoglyceride is phosphatidylcholine.

In some embodiments, the methods also include adding one or more fattyacids to the reaction mixture. Preferably the fatty acids include 8-24carbon atoms and 0-6 cis or trans double bonds with or without methylbranches and/or hydroxyl groups at any carbon atom.

In other embodiments, the methods also include adding one or more agentsselected from the group consisting of monoglyceride; diglyceride;polyglycerol fatty acid ester; sucrose fatty acid ester; sorbitan fattyacid ester; glycerol and other alcohol functional groups includingserine and ethanolamine; and solvents. Preferably the one or more agentsis monoglyceride. Preferred monoglycerides include monoglycerides havingan acyl group; the acyl group preferably includes 8-24 carbon atoms and0-6 cis or trans double bonds with or without methyl branches and/orhydroxyl groups at any carbon atom.

In still other embodiments, the methods include adding calcium ions orother multivalent ions.

According to another aspect of the invention, methods for makinglysophosphoglyceride are provided. The methods include contactingungranulated/solid matrix 3-sn-phosphoglyceride with phospholipase A₂ toform a reaction mixture, and recovering lysophosphoglyceride formed inthe reaction mixture.

In preferred embodiments, the reaction mixture further contains one ormore fatty acids. Preferably the fatty acids include 8-24 carbon atomsand 0-6 cis or trans double bonds with or without methyl branches and/orhydroxyl groups at any carbon atom.

In other preferred embodiments, the reaction mixture further contains anagent selected from the group consisting of monoglyceride; diglyceride;polyglycerol fatty acid ester; sucrose fatty acid ester; sorbitan fattyacid ester; glycerol and other alcohol functional groups includingserine and ethanolamine; and solvents. Preferably the agent ismonoglyceride. More preferably, the monoglyceride has an acyl group andthe acyl group comprises 8-24 carbon atoms and 0-6 cis or trans doublebonds with or without methyl branches and/or hydroxyl groups at anycarbon atom.

In certain of the foregoing methods for making lysophosphatidylcholine,the step of recovering comprises separating lysophosphoglyceride fromone or more reaction mixture constituents selected from the groupconsisting of 3-sn-phosphoglyceride, fatty acid, and the agent.

In some embodiments, the step of separation of lysophosphoglyceride fromthe one or more reaction mixture constituents comprises extraction withacetone.

In other embodiments, the 3-sn-phosphoglyceride in the reaction mixtureis greater than about 40% by weight of the mixture, greater than about50% by weight of the mixture, or greater than about 60% by weight of themixture.

According to still another aspect of the invention, lysophosphoglycerideproduced by the forgoing methods is provided.

In preferred embodiments of any of the foregoing claims, thelysophosphoglyceride is lysophosphatidylcholine, and/or the3-sn-phosphoglyceride is phosphatidylcholine.

Methods for making a composition containing lysophosphoglyceride,monoglyceride and fatty acid are provided according to another aspect ofthe invention. The methods include contacting a reaction mixture ofungranulated/solid matrix 3-sn-phosphoglyceride and monoglyceride withphospholipase A₂, and recovering a lipid complex containinglysophosphoglyceride, monoglyceride and fatty acid. The molar ratio oflysophosphoglyceride to the sum of monoglyceride and fatty acid in therecovered lipid complex composition is between 1:3 and 1:12. Preferablythe molar ratio of lysophosphoglyceride to the sum of monoglyceride andfatty acid in the recovered lipid complex composition is between 1:5 and1:6.

In some embodiments the recovered lipid complex composition has alysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2and 1:2:4. Preferably the recovered lipid complex composition has alysophosphoglyceride:monoglyceride:fatty acid molar ratio selected fromthe group consisting of 1:4:2, 1:3:3 and 1:3:2.

In certain embodiments the monoglyceride is derived from naturaltriglyceride. In other embodiments, the step of recovering the lipidcomplex comprises removal of water.

In preferred embodiments of the foregoing methods, thelysophosphoglyceride is lysophosphatidylcholine.

According to yet another aspect of the invention, drug deliverycompositions are provided. The drug delivery compositions include alipid matrix, at least part of which is in a lamellar phase, and apharmaceutically acceptable carrier.

According to a further aspect of the invention, other drug deliverycompositions are provided. The drug delivery compositions include alipid matrix, at least part of which is in a hexagonal phase or aninverse hexagonal phase, and a pharmaceutically acceptable carrier.

According to a still another aspect of the invention, additional drugdelivery compositions are provided. The drug delivery compositionsinclude a lipid matrix, at least part of which is in a phase other thana lamellar phase, hexagonal phase or inverse hexagonal phase, and apharmaceutically acceptable carrier.

In certain embodiments of the foregoing drug delivery compositions, thelipid matrix includes from about 0 to about 8 moles of water per mole oflipid. In some preferred embodiments, the lipid matrix includes at leastabout 1 mole of water per mole of lipid. In other preferred embodiments,the lipid matrix includes at least about 3 moles of water per mole oflipid. In still other preferred embodiments, the lipid matrix includesat least about 8 moles of water per mole of lipid.

In other embodiments of the foregoing drug delivery compositions, thelipid matrix comprises lysophosphoglyceride, monoglyceride and fattyacid, and the molar ratio of lysophosphoglyceride to the sum ofmonoglyceride and fatty acid in the lipid matrix is between 1:3 and1:12. Preferably the molar ratio of lysophosphoglyceride to the sum ofmonoglyceride and fatty acid in the lipid matrix is between 1:5 and 1:6.In certain preferred embodiments, the lipid matrix has alysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2and 1:2:4. More preferably, the lipid matrix has alysophosphoglyceride:monoglyceride:fatty acid molar ratio selected fromthe group consisting of 1:4:2, 1:3:3 and 1:3:2.

In some preferred embodiments of the foregoing drug deliverycompositions, the lysophosphoglyceride is lysophosphatidylcholine. Inother embodiments, the drug delivery compositions also include one ormore water soluble or water insoluble pharmaceutical compounds.

According to still another aspect of the invention, a lipid matrix isprovided. The lipid matrix includes lysophosphatidycholine,monoglyceride and fatty acids, and has a viscosity indicative of anon-Newtonian fluid. In embodiments in which the lipid matrix includeswater, the molar ratio of water:lipid matrix preferably is less than orequal to about 8:1.

In another aspect, the invention provides methods for making acomestible lipid matrix composition in a reactor vessel. The methodsinclude preparing a lipid matrix containing lysophosphatidylcholine,monoglyceride and fatty acids, adding a dilute aqueous acid to the lipidmatrix in the reactor vessel, mixing and heating the reactor vesselcontents to prepare a protonated aqueous lipid matrix, combining theprotonated aqueous lipid matrix with comestible components in a reactorvessel, and mixing the comestible components and the protonated aqueouslipid matrix in the reactor vessel to make a comestible lipid matrixcomposition. In some embodiments, about 8 moles of water are added permole of lipid matrix. In other embodiments, the reactor vessel contentsare heated to about 50-60° C. In yet other embodiments, the comestiblecomponents include compounds selected from the group consisting ofprotein, sugar and starch.

According to another aspect of the invention, methods for treatingcystic fibrosis are provided. The methods include administering to asubject in need of such treatment an effective amount of any of theforegoing compositions. In preferred embodiments, a physiologicalparameter of the subject related to the cystic fibrosis is improved.Preferred physiological parameters include height-for-age Z score,weight-for-age Z score, forced expiratory volume, energy intake fromdiet, essential fatty acid status, fat soluble vitamin status andretinol binding protein status.

According to still another aspect of the invention, nutritionalsupplements are provided. The nutritional supplements include aneffective amount of any of the foregoing compositions. In preferredembodiments, the nutritional supplement are used for the treatment ofcystic fibrosis. Use of any of the foregoing compositions in thepreparation of medicaments, particularly for treatment of cysticfibrosis, also is provided.

These and other aspects and objects of the invention will be describedin further detail in connection with the Detailed Description of theInvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the composition over time of the phosphatidylcholinehydrolysis reaction mixture without added monoolein.

FIG. 2 depicts the composition over time of the phosphatidylcholinehydrolysis reaction mixture with monoolein added at a 1:3 molar ratio.

FIG. 3 shows the birefringence characteristics of phosphatidylcholine,phosphatidylethanolamine and basic neat lipid matrix.

FIG. 4 and FIG. 5 depicts non-Newtonian flow behavior characteristics ofa basic neat lipid matrix containing <1% moisture (FIG. 5 showsmeasurements at 50° C.).

FIG. 6 shows differential scanning calorimetry of varying mole ratios oflipid matrix.

FIG. 7 depicts differential scanning calorimetry analysis of lipidmatrix in aqueous media.

FIG. 8 shows viscosity of LYM-X-SORB™ with increasing amounts of water.

FIG. 9 shows schematic representations of a lipid matrix in lamellarphase (FIG. 9A), hexagonal phase (FIG. 9B) and inverse hexagonal phase(FIG. 9C).

FIG. 10 depicts the structural rearrangement from lamellar phase tohexagonal phase.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes improved methods for makinglysophosphatidylcholine which involve hydrolyzing ungranulated/solidmatrix phosphatidylcholine by contacting it with a phospholipase,preferably phospholipase A₂, in a reaction mixture. Phospholipase A₂ ispreferred because the lysophosphatidylcholine produced by hydrolysis ofphosphatidylcholine at the 2-position is the biologically preferredlysophosphatidylcholine (in contrast with lysophosphatidylcholineproduced by hydrolysis of phosphatidylcholine at the 1-position). Inbroader aspects of the invention, methods of modifying3-sn-phosphoglycerides are provided. In such methods, an amount ofphospholipase that is sufficient to modify one or more ester bondlinkages (designated A, B, C, and D) of the 3-sn-phosphoglyceridemolecules is combined with ungranulated/solid matrix3-sn-phosphoglyceride. The preferred embodiments described herein,pertaining to the hydrolysis of phosphatidylcholine by phospholipase A2,are exemplary of the more general methods provided by the invention.

In certain aspects of the methods, the reaction mixture can includefatty acids and/or an agent. The formed lysophosphatidylcholineoptionally may be separated from the added agent and/or the fatty acidswhich are liberated by the action of phospholipase A₂ onphosphatidylcholine. The method enables substantially completehydrolysis of inexpensive ungranulated/solid matrix phosphatidylcholineto lysophosphatidylcholine in a single step. If desired, the agent maybe monoglyceride and the resulting lipid matrix oflysophosphatidylcholine, monoglyceride and fatty acids may be separatedfrom the phospholipase A₂, trace unreacted phosphatidylcholine, water,organic solvents, and any impurities present in the reaction mixture.The resulting lipid matrix, in defined molar ratios, is useful as acaloric medical food to provide both polyunsaturated fatty acids and theabsorbable form of choline (lysophosphatidylcholine), and is useful as adrug delivery system.

The following abbreviations are used herein for components of thedescribed methods and lipid matrices: phosphatidylcholine (PC),lysophosphatidylcholine (LPC), monoglyceride (MG), fatty acids (FA) andphospholipase A₂ (PLA₂).

The starting material for the method is phosphatidylcholine, aphospholipid composed of a polar hydrophilic head group of choline,phosphate and glycerol linked to a nonpolar hydrophobic tail groupconsisting of two fatty acid molecules. Phosphatidylcholine may beobtained with specific fatty acid groups, or with a mixture of variousfatty acid groups.

One of the advantages of the presently disclosed methods is the abilityto use ungranulated/solid matrix phosphatidylcholine as a startingmaterial. This kind of phosphatidylcholine, although significantly lessexpensive than purified, granulated phosphatidylcholine used in previousenzymatic methods of phosphatidylcholine hydrolysis, has not previouslybeen used to make lysophosphatidylcholine because it was thought to beimpossible to hydrolyze efficiently due to the small surface area of asolid matrix (e.g., block) of phosphatidylcholine as compared to thecombined surface area of the individual granules of granulatedphosphatidylcholine.

Previous methods of phosphatidylcholine hydrolysis (e.g., U.S. Pat. No.5,716,814 to Yesair) hydrolyzed an aqueous dispersion of granulatedphosphatidylcholine. In contrast, the present disclosure demonstrates,unexpectedly, that the hydrolysis of a solid block ofphosphatidylcholine can be successfully accomplished. Even moresurprisingly, it now has been determined that an economicalungranulated/solid matrix phosphatidylcholine can be hydrolyzed asefficiently as a more expensive granulated preparation using anefficient mixing apparatus (e.g., Littleford-Day reactor). Examples ofungranulated/solid matrix phosphatidylcholine is Nattermann 8729,Phospholipon® 80 and Phospholipon® 90, all of which are soyphosphatidylcholine preparations.

As disclosed herein, a reaction mixture of phosphatidylcholine andphospholipase A₂ is prepared by combining these reaction components. Theterm “mixture” merely indicates that the components are in contact withone another.

Other components can be added to the reaction mixture, includingagent(s) (e.g., monoglyceride(s)), fatty acid(s), multivalent ions(e.g., calcium), buffer salts (e.g., sodium bicarbonate), acids orbases, and water.

The addition of an agent is believed to achieve several purposes. First,the molecules of the agent are believed to separate thephosphatidylcholine molecules to allow greater access to thephosphatidylcholine by phospholipase A₂, thus enabling completehydrolysis to lysophosphatidylcholine. Second, addition of the agent isbelieved to maintain the proper structure during the hydrolysisreaction, i.e., polar head group associated with the bulk water phasecontaining enzyme. Third, addition of the agent is believed to maintainfluidity of the phosphatidylcholine bilayer to enhance hydrolysis byphospholipase A₂. Fourth, addition of the agent is believed to removethe hydrolytic products (LPC and FA) from the surface of theungranulated/solid matrix. Thus, any agent which has one or more of theaforementioned characteristics is believed suitable for adding tophosphatidylcholine to facilitate hydrolysis by phospholipase A₂. It ispreferable that the agent be selected from amongst the group consistingof monoglyceride, diglyceride, polyglycerol fatty acid ester, sucrosefatty acid ester, sorbitan fatty acid ester, glycerol and other alcoholfunctional groups. Most preferably, the agent is monoglyceride.

Monoglyceride is composed of a glycerol head group to which one fattyacid acyl group is attached. Preferred acyl groups of monoglycerideuseful in the invention may include 8-24 carbon atoms and 0-6 cis ortrans double bonds with or without methyl branches and/or hydroxylgroups at any carbon atom. Acyl groups of monoglyceride preferablyinclude 1-4 double bonds in the carbon chain. The monoglyceride may behighly purified or may be added in a crude form, depending on the needsof the user and the tolerance for impurities in the reaction mixture.Monoglycerides useful in the invention may represent a mixture ofmonoglyceride molecules having different size and saturation-state acylgroups, or the monoglyceride may represent only a single type of acylgroup, e.g., mono-olein, mono-palmitin. Examples of a mixture ofmonoglycerides useful in the invention include Dimodan™ LSK, Dimodan™ OKand flaxseed oil monoglycerides (Danisco Cultor, New Century, Kans.).

Diglyceride molecules are also useful in the method of the invention forenhancing the hydrolysis of phosphatidylcholine by phospholipase A₂. Adiglyceride molecule consists of a glycerol head group to which twofatty acid acyl groups are attached. As with the acyl group ofmonoglyceride, the acyl groups of diglyceride can include 8-24 carbonatoms and 0-6 cis or trans double bonds with or without methyl branchesand/or hydroxyl groups at any carbon atom. The acyl groups ofdiglyceride preferably have carbon chain links from 8 to 22 carbon atomsand 1 to 4 unsaturations. As with monoglyceride, the specific acylgroups, purity, and mixture of diglyceride molecules useful in theinvention depend on the requirements of the individual user. Anycombination or type of diglyceride molecules is contemplated by theinvention, so long as the hydrolysis of phosphatidylcholine is enhanced.

Other agents such as polyglycerol fatty acid esters, sorbitan fatty acidesters, sucrose fatty acid esters and glycerol may also enhance thehydrolysis of phosphatidylcholine by removing the hydrolytic productsfrom the solid PC surface and by modulating the surface structure and/orfluidity of the solid PC. Such compounds are described in U.S. Pat. No.4,849,132. A polyglycerol fatty acid ester molecule consists of mono-,di- or polyesters of fatty acids with 4-12 polymerized glycerolmolecules. A sorbitan fatty acid ester molecule consists on mono-, di-or polyesters of fatty acids with sorbitol, sorbitan and sorbide. Asucrose fatty acid ester molecule consists of mono-, di- or polyestersof fatty acids with sucrose. As with the acyl group of monoglyceride,the fatty acids/acyl groups of polyglycerol fatty acid ester, sorbitanfatty acid ester and sucrose fatty acid ester can include 8-24 carbonatoms and 0-6 cis or trans double bonds with or without methyl branchesand/or hydroxyl groups at any carbon atom. Preferably these acyl groupshave carbon chains of 8-22 carbon atoms and 1-4 unsaturations. As above,the specific acyl groups, purity, and mixture of agent molecules usefulin the invention depend on the requirements of the individual user.

Any single agent or mixture of different agents which enhanceshydrolysis of phosphatidylcholine is contemplated as useful for theinvention. The aforementioned agents are available commercially from avariety of sources.

Phosphatidylcholine is hydrolyzed to lysophosphatidylcholine by theaction of phospholipase A₂, which severs the ester bond linking a fattyacid group to the 2-position of the glycerol in the head group ofphosphatidylcholine. Phospholipase A₂ may be purified from a variety ofsources, or it may be obtained from commercial sources (e.g., Lecitase™,Novo Nordisk, Denmark). For full activity, phospholipase A₂ is believedto require the presence of Ca²⁺ ions in the reaction mixture. Whiletypically there is a low level of Ca²⁺ ions in the commercialphospholipase A₂ preparations such that phospholipase A₂ is active, itis preferred that Ca²⁺ ions be added to the reaction mixture for fullactivity. It should be noted that Ca²⁺ ions are depleted from thereaction mixture by ionic bonding with the acid group of fatty acidsliberated during hydrolysis of phosphatidylcholine. Therefore it ispreferred that sufficient Ca²⁺ ions are added to the reaction mixture tomaintain full activity of phospholipase A₂. In this invention, it ismost preferable that the user supplement the calcium ion concentrationto achieve a molar ratio of calcium ion:phosphatidylcholine of at least0.18:1.

Phospholipase A₂ catalyzes the hydrolysis of the 2-acyl bond of3-sn-phosphoglycerides. Accordingly, the present methods that utilizephospholipase A₂ in the hydrolysis of phosphatidylcholine can be usedfor hydrolysis of other 3-sn-phosphoglycerides. Adjustments in reactionconditions for specific 3-sn-phosphoglyceride molecules other thanphosphatidylcholine can be made in accordance with the known propertiesof phospholipases and/or with routine experimentation.

It will be recognized by persons of skill in the art that other ions maybe substituted for the Ca²⁺ ions in order to maintain full activity ofthe phospholipase A₂ enzyme. While not all ions may substitute for Ca²⁺ions in this reaction, the specific type and concentration of ionsadequate for maintenance of phospholipase A₂ activity may be testedusing routine methods by one of ordinary skill in the art.

As disclosed above, the method of making lysophosphatidylcholineincludes forming a reaction mixture of phosphatidylcholine andphospholipase A₂. In contrast to previous methods of making LPC, whichrequired granulation of PC and formation of an aqueous dispersion of thePC in water, the present methods do not require PC granulation.

Other reaction conditions, such as pH, time and temperature, may bevaried to achieve optimal hydrolysis of phosphatidylcholine. Forexample, phospholipase A₂ has a pH optimum of pH 8-9 which should bemaintained to retain maximal enzyme activity. During the progress of thereaction, as fatty acids are released by hydrolysis ofphosphatidylcholine, the pH of the reaction mixture may change. Such achange of pH may require the addition of base to maintain the optimalrange of pH 8-9. Any base which effectively raises the pH to the optimalrange without interfering with the hydrolysis of phosphatidylcholine maybe used. Aqueous sodium hydroxide or sodium bicarbonate may be used forthis purpose. Other formulations of sodium hydroxide or other bases maybe employed for the same purpose. If the particular reaction conditionsemployed result in an increase in pH, then it is contemplated that acidmay be added to maintain optimal pH.

Hydrolysis of 3-sn-phosphoglycerides (e.g., phosphatidylcholine) byphospholipase A₂ will proceed at many temperatures less than or equal toabout 80° C., such as at the optimal temperature for phospholipase A₂enzymatic activity (70-80° C.), but unexpectedly it was determined thatthe preferred reaction temperature is from about 50° C. to about 55° C.Other suitable temperatures may be determined with routineexperimentation by one of ordinary skill in the art depending on thespecific reaction mixture employed in the phosphoglyceride hydrolysisreaction.

The time for the reaction may be chosen by the user of the method as isconvenient, so long as the hydrolysis of phosphatidylcholine hasprogressed to an extent desired. It should be noted that the previousmethods of PC hydrolysis by phospholipase A₂ proceeded to completeconversion of PC to LPC in several days. In the presently disclosedmethods, the hydrolysis of PC can be completed in several hours. Thisreduction in reaction time is entirely unexpected in view of thesignificantly lesser surface area of the ungranulated solid matrix PCused in the reaction to produce LPC as compared to the surface area ofgranulated PC materials used in previous methods. It is preferred thatthe reaction proceed for 24 hours or less, more preferably for less than12 hours, still more preferably for less than 6 hours, and mostpreferably for less than 4 hours.

The amount of phosphatidylcholine to be used in the method of theinvention is quantified as a weight percentage of the total solids inthe reaction mixture. Weight percentage is calculated by dividing theweight of a single reaction component divided by the sum of the weightsof all solid components in the reaction mixture. Previous methods ofhydrolyzing phosphatidylcholine to produce LPC, which favored reactionmixtures comprising less than about 40% phosphatidylcholine by weightdue to the likelihood that reaction mixtures with greater percentages ofPC were likely to separate into a non-lamellar two-phase system whichdoes not permit efficient hydrolysis of the phosphatidylcholine. Incontrast the present methods, which provide hydrolysis of ungranulated,solid matrix PC to produce LPC, can accommodate weight percentages of PCthat are greater than or equal to 40% of the total solids in thereaction mixture.

Preferably the reaction mixture is formed in a reactor device thatmixes, stirs, and/or heats the reaction mixture. The mixing and stirringof the reaction mixture is believed to bring the phospholipase A₂ (andother reaction components) into contact with the solid matrix ofphosphatidylcholine, thereby increasing the efficiency of the hydrolysisreaction and reducing the time required for complete hydrolysis. Oneexample of a suitable reactor device is the Model M5 (½ HP motor) mixerof Littleford Day Inc. (Florence, Ky.). This mixture has a 5 litercapacity. Other similar reactors having larger capacity can be used toscale up the reaction.

The reactors also can provide heating and drying functions. The reactionmixture can be heated to optimal reaction temperatures, which typicallydepend on the temperature optimum of the enzyme. Heating also can beemployed to dry the reaction products after completion of the hydrolysisreaction. Reactors can be equipped with other functions to aid drying,such as vacuum. The reactor can be used to modify the mole ratios of MGand FA to LPC and can be used to change the molecular structure betweenlamellar and inverse hexagonal organization(s) or combinations thereof.Using the reactor, the inverse hexagonal structure of LXS™ can beformulated with protein, carbohydrate, starch and flavors to produce apowdered formulation of LXS™. The reactor may also be used to form adrug/LYM-X-SORB™ inclusion complex.

An agent may be added to the mixture at any weight percentage whichenhances the hydrolysis of phosphatidylcholine over the amount ofhydrolysis of phosphatidylcholine alone by phospholipase A₂. Mostpreferably, the agent is monoglyceride. When present in the reactionmixture, virtually any amount of monoglyceride will enhance thehydrolysis of phosphatidylcholine by phospholipase A₂. Preferably themolar ratio of phosphatidylcholine:monoglyceride is 1:0.1-1:10. To reachhigh yields of lysophosphatidylcholine it is preferred to have a molarratio of phosphatidylcholine:monoglyceride of about 1:1-1:5. Mostpreferably, the molar ratio of phosphatidylcholine:monoglyceride isabout 1:3.

The desired end products of the reaction of phosphatidylcholine andagent with phospholipase A₂ are lysophosphatidylcholine alone, acombination of lysophosphatidylcholine with fatty acid or agent, orlysophosphatidylcholine in combination with fatty acid and agent. Inparticular, when the agent is monoglyceride, a preferred end product isa lipid matrix comprising lysophosphatidylcholine, monoglyceride andfatty acid. The utility of this lipid matrix has been disclosed, forexample, in U.S. Pat. Nos. 4,874,795 5,314,921 and 5,972,911.

Where the end product is a lipid matrix composition oflysophosphatidylcholine, monoglyceride and fatty acid, it is preferredthat the constituents of the lipid matrix be present in the molar ratioof lysophosphatidylcholine:the sum of monoglyceride and fatty acid ofabout 1:3 to 1:12. Most preferably, the molar ratio oflysophosphatidylcholine:the sum of monoglyceride and fatty acid is about1:5-1:6. It is also preferred that the individual components of thelipid matrix are present in particular molar ratios in relation to oneanother. Thus, it is preferred that the molar ratios oflysophosphatidylcholine:monoglyceride:fatty acid are 1:4:2-1:2:4. Mostpreferably, the molar ratios oflysophosphatidylcholine:monoglyceride:fatty acid are either 1:4:2, 1:3:3or 1:3:2.

Additional monoglycerides and fatty acids may be added to thelysophosphatidylcholine/monoglyceride/fatty acid mixture and melted ormixed to yield compositions of matter as defined in U.S. Pat. No.4,874,795. Thus, monoglyceride and/or fatty acid may be added to thelipid matrix if it is desired to alter the molar ratios of monoglycerideand/or fatty acid to yield a desired product.

The lipid matrix produced by the method of the invention is useful for,inter alia, delivery of drugs. When so desired, a pharmaceuticalcomposition may be added to the reaction mixture, for inclusion in thelipid matrix, at any time which does not adversely affect the integrityof the pharmaceutical composition. Preferably the desired pharmaceuticalcomposition is added subsequent to the formation of the lipid matrixand/or during the transition between the lamellar and inverse hexagonalorganization(s).

Preferably the methods disclosed herein include a step of recoveringfrom the reaction mixture the lysophosphatidylcholine formed in thereaction mixture. As used herein, “recovering” means recovering thelysophosphatidylcholine from one or more of the components of thereaction mixture. The actual form of the lysophosphatidylcholine canvary, i.e., the lysophosphatidylcholine recovered can be recoveredcomplexed with other components of the reaction mixture. For example,recovering lysophosphatidylcholine includes recovering a lipid complexwhich contains lysophosphatidylcholine, fatty acid and agent. Recoveringlysophosphatidylcholine also embraces recovering lipid complexes whichcontain lysophosphatidylcholine and agent or lysophosphatidylcholine andfatty acid. It is not necessary that the lysophosphatidylcholine orlysophosphatidylcholine-containing lipid complex be purified to beconsidered recovered. Therefore, the lysophosphatidylcholine orlysophosphatidylcholine-containing lipid complex can contain otherconstituents present in the reaction mixture, such as Ca²⁺ orphospholipase A₂. The lysophosphatidylcholine, however, when “recovered”is sufficiently isolated from other materials so as to be useful as anisolate of lysophosphatidylcholine or of alysophosphatidylcholine-containing lipid complex. Thelysophosphatidylcholine or lysophosphatidylcholine-containing lipidcomplex which is recovered can, however, be purified if so desired.

The step of recovering can include one or more process steps wherebylysophosphatidylcholine is separated from one or more of theconstituents of the reaction mixture. Thus, lysophosphatidylcholine maybe separated from fatty acid, agent (e.g. monoglyceride) or fatty acidand agent. Separation includes separation of the desiredlysophosphatidylcholine or lysophosphatidylcholine-containing lipidcomplex from the reaction mixture as well as separation of an unwantedreaction component from the reaction mixture. For example, the reactionmixture can be extracted with acetone to preferentially separatelysophosphatidylcholine from other reaction mixture components, asdescribed herein. In other embodiments, where a lipid matrix comprisinglysophosphatidylcholine, monoglyceride and fatty acid is the desired endproduct, other reaction components such as phospholipase A₂, water,organic solvents and excesses of monoglyceride or fatty acid can beseparated from the lipid matrix. Alternatively, water can be separatedfrom other reaction mixture components by heating or drying the reactionmixture as is described herein. Other methods of separating selectedproducts of the enzymatic hydrolysis of phosphatidylcholine are providedherein, and still others will be known to one of ordinary skill in theart.

Many methods known to those of ordinary skill in the art will beapplicable to separation of lysophosphatidylcholine from other reactioncomponents based on differential solubilities, molecular weights,molecular sizes or other properties. For example,lysophosphatidylcholine may be separated from other components bypreparative chromatography. Preferably, lysophosphatidylcholine can beseparated by extraction of the reaction mixture with acetone. Thismethod relies on the insolubility of phospholipids in acetone;lysophosphatidylcholine precipitates as a solid which is easilyrecovered from other reaction constituents. Other separation methodswill be known to those of ordinary skill in the art.

Compositions containing lysophosphatidylcholine, alone or in combinationwith monoglyceride and/or fatty acids, are useful as emulsifiers,antioxidants and surfactants in cosmetic and dermatologicalpreparations.

As disclosed above, the methods of the invention also contemplate theremoval of water and/or other solvents from the reaction mixture torecover desired end products. Thus, the method of making any of theforegoing products may include the removal of water or solvents as partof, or separate from, the separation processes outline above.

Any method known in the art for the removal of water, aqueous solvents,or mixtures of aqueous and organic solvents may be used so long as thedesired end products of the hydrolysis reaction are not adverselyaffected. It is preferred that methods which are scalable to industrialproduction of lysophosphatidylcholine or lipid matrix compositions beemployed. For example, solvents may be removed from desired end productsby heating, vacuum, lyophilization, or spray drying processes. Suchmethods may be employed for such a time and to such an extent so as toremove all or part of the water or solvent mixtures as desired by theuser. Preferably, reaction products are heated to remove water, therebyyielding a paste of lysophosphatidylcholine or lipid matrix.

In another aspect of the invention, a lipid matrix having certainphysical properties is provided. Upon addition of water to a neat lipidmatrix of lysophosphatidylcholine, monoglyceride and fatty acid withheating, the physical properties of the matrix change to includeincreased viscosity and altered X-ray diffraction patterns. At a molarratio of 8 moles water per mole of lipid matrix, all of the lipid matrixbecomes converted to the new form. As described in the Examples, thedata are consistent with conversion of a lamellar bilayer structure to ahexagonal structure with the addition of water and heat. The X-ray datafavors an inverse hexagonal structure, with the matrix organized to haveits polar hydrophilic region on the interior of matrix molecules. Thedata is also consistent, however, with a hexagonal structure wherein thematrix is organized to have its polar hydrophilic region on the exteriorof matrix molecules (see FIGS. 9 and 10).

Thus the invention includes methods for making hexagonal phase lipidmatrices by contacting a neat lipid matrix with water and heat.Preferably the matrix is mixed during or after the addition of water. Inpreferred methods, one or more pharmaceutical compounds are included inpreparation of the hexagonal phase matrix, such as by adding thecompounds during the addition of water to the neat lipid matrix.Alternatively, the pharmaceutical compounds can be added after theformation of the hexagonal matrix is complete, optionally after theformation of particles of desired size (e.g., by sonication in thepresence of bicarbonate or other suitable ions as known to one ofordinary skill in the art).

One feature of the hexagonal matrix so formed is that it can be used asa carrier or delivery vehicle for pharmaceuticals that are hydrophobicand/or hydrophilic. The hexagonal matrix is believed to be particularlysuited for delivery of nucleic acids and vaccine constituents. Forexample, a delivery vehicle for hydrophobic and hydrophilicpharmaceuticals can be prepared by including a hydrophilic compoundduring the preparation of the hexagonal lipid matrix particles, wherebythe hydrophilic compounds become incorporated into the interior ofinverse hexagonal lipid matrix particles, interacting with or binding tothe polar hydrophilic region. Hydrophobic compounds can then be added toassociate with or bind to the outside of the inverse hexagonal lipidmatrix particles (the nonpolar hydrophobic region of the lipid matrix).If non-inverse hexagonal particles are formed, then the order ofaddition of hydrophobic and hydrophilic compounds may be reversed.

Another use of the compositions described herein is as nutritionalsupplements. As will be known to one of ordinary skill in the art, lipidmatrices have been used for nutritional supplements, particularly tosupplement the diet of subjects having particular disorders that requireadditional nutrition, such as wasting diseases, cancer and cysticfibrosis. One recent study has shown that a lipid matrix can be usedeffectively in the treatment of cystic fibrosis patients (Lepage et al.,J. Pediatr. 141:178-185, 2002, incorporated by reference). Thus, thepresent invention includes the use of the compositions described hereinfor the treatment of disease by improving the nutritional status ofpatients, using methods as are known to one of ordinary skill in theart, such as the Lepage reference.

In particular, methods for treating cystic fibrosis are provided. Themethods include administering to a subject in need of such treatment aneffective amount of the compositions described herein. The treatmentsfavorable affect a physiological parameter of the subject related to thecystic fibrosis. Preferred physiological parameters includeheight-for-age Z score, weight-for-age Z score, forced expiratoryvolume, energy intake from diet, essential fatty acid status, fatsoluble vitamin status and retinol binding protein status. Thus theinvention also provides nutritional supplements that include aneffective amount of any of the foregoing compositions. The nutritionalsupplements can be formulated according to standard methodology in thepharmaceutical and nutritional arts, for example as described in Example3 below.

EXAMPLES Example 1 Preparation of Lysophosphatidylcholine and LipidMatrix Compositions Hydrolysis of Solid Phosphatidylcholine

Briefly, using a more economical, solid matrix, ungranulated form of soyPC (Nattermann 8729, Nattermann Aventis Pharma Deutschland GmbH,Cologne, Germany) and an efficient mixing apparatus (Littleford/Dayreactor, Model M5, ½ HP motor, 5 L capacity, Littleford Day Inc.,Florence, Ky.), the hydrolysis of PC to lysophosphatidylcholine (LPC)using phospholipase A₂ and monoglyceride (MG) was complete (>99%) within5-6 hours. In the following experiments 400 g of Nattermann 8729 werehydrolyzed in a 5 L Littleford/Day reactor. The load phospholipid,monoglyceride, phospholipase A₂ enzyme, buffer and water representedapproximately 40% of the 5 liter capacity of the reactor.

In the hydrolysis of phosphatidylcholine (PC) using the enzymephospholipase A₂ (PLA₂) (Lecitase, Novo Nordisk, Denmark), approximately300,000 Lecitase Units (one unit produces 1 μmole of fatty acid perminute) were used to hydrolyze 400 g of Nattermann 8729 in the presenceof monoglycerides (MG) (Dimodan™ LSK and Dimodan™ OK, Danisco Cultor,New Century, Kans.). The initial mole ratio of the sum of PC+LPC:MG was1:3. Time for complete hydrolysis was 4 to 5 hours (batch 5.06; seeTable 1). As shown in FIG. 1, PC hydrolysis was present at 4 hours andabsent at 5 hours. Also note that the moles of fatty acids (FA) werealways higher than the corresponding moles of lysophosphatidylcholine(LPC) when in fact they should be equivalent as one mole of PC ishydrolyzed to 1 mole of LPC and 1 mole of FA (1 PC→1 LPC+1 FA). Reactionproducts were determined by HPLC analysis.

Based upon the addition of reactants, the final lipid matrix preparationshould have a theoretical mole ratio of 1:4:2 (LPC:MG:FA); whereas, infact both the MG and FA were greater in the tested preparations. Asshown in FIG. 2, the mole ratio of MG exceeded 4 and FA also exceededits theoretical value of 2. In calculating these results theconcentration of LPC was the base of 1. Batches 5.19 and 5.20 (bothanalyzed in duplicate), and batches CS-1, CS-2 and CS-3 contained thesame level of enzyme and reactants as noted above.

To explain the increased mole ratios of both MG and FA, a loss of LPCwas hypothesized to occur during the hydrolysis of PC to yieldglycerylphosphorylcholine (GPC). This could occur by inappropriatehydrolysis of 2 fatty acids from PC, or by hydrolysis of a fatty acidfrom LPC, as follows: 1 PC→GPC+2 FA or 1 LPC→1 GPC+1 FA.

Effect of Fatty Acid Addition to Reaction Mixture

In the hydrolysis of PC using PLA₂, the maximum rate of hydrolysis isdelayed and this delayed time (tau) can be reduced by adding both LPCand FA (Bent and Bell, Biochimica et Biophysica Acta 1254:349-360;1995).

In Nattermann 8729, there is about 4.5% LPC but no FA. Therefore, theeffect of adding fatty acids to the initial reaction mixture was tested.

GPC production was evaluated in four batches without any added FA(5.21A, 5.21B, 5.23, 5.24). Batch 5.21A was a control in which unalteredhydrolysis was performed as described above. This batch was vacuum driedto 1.34% water. Batch 5.21B was like 5.21.A, except that calcium wasadded (0.5 moles) and the amount of water was increased to 1.95% (±2moles). Batch 5.23 was like 5.21.A, except that 40 ml concentrated HClwas added after PC hydrolysis; this batch was vacuum dried to 0.67%water. Batch 5.24 was like 5.21.A, except that 40 ml concentrated HClwas added after PC hydrolysis; this batch was vacuum dried to about1.70% water (±2 moles).

The amounts of GPC in the four batches was analyzed by phosphorous NMR.Samples 5.21A and 5.21B contained 16.67 and 19.80 mole % of GPC andsamples 5.23 and 5.24 contained 8.96 and 9.93 mole % of GPC. Therefore,the addition of acid (HCl) decreased the amount of GPC in the batches byabout 50%.

In contrast, the addition of 15 mole % of fatty acid (0.09 moles) to theinitial reaction mixture prior to hydrolysis (batch 5.25) showed zeroyield of GPC. Surprisingly, the complete hydrolysis of PC as analyzed byHPLC (using 300,000 units of enzyme) occurred within 2 hours withoutproduction of GPC (as analyzed by phosphorus NMR). Thus, the addition offatty acid both reduced the production of GPC as well as decreased thetime for complete hydrolysis of PC, which it believed to correspond tothe reduction of tau.

In these initial studies, approximately 300,000 units of PLA₂ were usedand there is a need to reduce the cost of the PC hydrolysis and lipidmatrix preparation. Therefore, decreased enzyme levels were evaluated inhydrolysis reactions as described above.

In batch 5.27, 150,000 units of PLA₂ enzyme (without adding FA to thereaction mixture) showed complete hydrolysis of PC within 5.5 hours.Using only 72,000 units of PLA₂ (batch 5.30, without FA) PC hydrolysiswas complete within 6-7 hours.

The addition of fatty acid reduced the time for complete hydrolysis ofPC. Using 150,000 units of PLA₂ with added FA, the hydrolysis of PCrequired approximately 2.75-3 hours (batches 5.28 and 5.29) in contrastto 5.5 hours, noted above for batch 5.27. Using 120,000 units of enzymewith added FA, hydrolysis was complete within 3-4 hours (batches 5.33and 5.32).

Therefore, the units of PLA₂ can be significantly reduced withoutdrastic effects on the reaction time. The effect on reaction time isreduced by the addition of fatty acids.

Effect of Mixing on Reaction

A newer Littleford/Day 5 liter reactor (Model M5, 1 HP motor) that has amore powerful motor was tested under the same reaction conditionsdescribed above. After the first batch using a 40% load it was apparentthat the maximum reaction capacity of 70% load would be possible.

Using the new 5 liter reactor with a 40% load, approximately 120,000units of PLA₂ enzyme and added FA, the hydrolysis of PC was completewithin 2.25 hours (batch 5.34) which contrasts with 3-4 hours notedabove for the old reactor.

Using the same reactor with a 70% load and proportionately the same120,000 units of PLA₂ and added FA, the hydrolysis of PC was completewithin the same time, 2.25 hours (batches 5.35 and 5.37).

In summary, the addition of fatty acids (FA) to the initial reactionmixture increased the yield of lysophosphatidylcholine (LPC) and reducedthe time required for complete hydrolysis of phosphatidylcholine (PC).The increased efficiency of the PLA₂ enzyme with added FA also permitsthe use of decreased enzyme concentrations for the hydrolysis of PC.Efficiency of mixing as gauged by the use of a more powerful mixer alsocontributed to reduced reaction time, regardless of the load status (upto maximum fill capacity; i.e., 70% for Model M5) of the mixer.

TABLE 1 ADDED ENZYME TIME-HR GPC BATCH # FA UNITS 100% HYDROL. MOLE % ½HP REACTOR 5.06 NONE 300,000 4 TO 5 ND 5.27 NONE 150,000 ca. 5.5  ND5.30 NONE 72,000 6 TO 7 ND   5.21A NONE 300,000 4 TO 5 16.67   5.21B19.80 5.23 NONE 300,000 4 TO 5 8.96 5.24 NONE 300,000 4 TO 5 9.93 5.25YES 300,000 2 0.00 5.28 YES 150,000 2.75 TO 3   ND 5.29 YES 150,000 2.75TO 3   ND 5.32 YES 120,000 3 TO 4 ND 5.33 YES 120,000 3 TO 4 ND 1 HPREACTOR 5.34 YES 120,000 ca. 2.25 ND 5.35 YES 120,000 ca. 2.25 ND 5.37YES 120,000 ca. 2.25 ND ND = Not determined

Example 2 Analysis of Physical Properties of Lipid Matrix PreparationsBirefringent and Rheological Characteristics

Example 1 shows a modification of the method to hydrolyzephosphatidylcholine (PC) and to produce an organized lipid matrix of PC,monoglyceride (MG) and fatty acids (FA). Using the same Littleford/Dayreactors [Model M5, ½ HP and 1 HP motors], the 30% water content of thereaction was removed with vacuum and heat within 16-18 hours. Theresulting basic neat lipid matrix, when viewed in a polarizing lightmicroscope, exhibited a unique birefringence of unknown structure thatwas different from the lamellar and hexagonal phases ofphosphatidylcholine and phosphatidylethanolamine, respectively (FIG. 3),but may be similar to the birefringent liquid crystalline phases inhuman intestinal contents during fat digestion (Holt, Fairchild andWeiss, Lipids 21:444-446, 1986).

Viscosity is the measure of the internal friction of a fluid. As shownin FIGS. 4 and 5, the basic neat lipid matrix containing <1% moistureshows non-Newtonian flow behavior characteristics. A dilatant fluidshows “shear-thickening” flow behavior; i.e., increasing viscosity withan increase in shear rate.

Differential Scanning Calorimetry (DSC)

Based upon differential scanning calorimetry (DSC) analysis of apalmitoyl lipid matrix in the presence of excess MG and FA, one mole ofLPC will interact with a maximum of 5-6 moles of MG/FA (FIG. 6); i.e.,one mole of LPC will form a complex with 3 moles of MG and 3 moles ofFA.

Sonication of the protonated lipid matrix yields particles having a size˜150 nm; whereas, sonication of the ionized lipid matrix yields ˜70 nmparticles (see U.S. Pat. No. 5,891,466 to Yesair). The DSC analysis of apalmitoyl (16:0) matrix [16:0 LPC (1 mole); 16:0 MG (3 moles); 16:0 FA(3 moles)] that was sonicated in water showed a melting point at 64.6°C., the ionized lipid matrix in 15 mM sodium bicarbonate melted at 52°C. and the ionized lipid matrix containing 0.5 moles of calcium ions permole of the monomeric lipid matrix had a broad melting peak at about35-40° C. (FIG. 7). Greater amounts of calcium ions had no furthereffects.

Viscosity with Variable Water Content

As shown previously, the proposed organization of the lipid matrix hasboth a polar region and a non-polar hydrophobic region. The formercontains phosphorylcholine of LPC, carboxylic acid of FA and glycerol ofMG. The space between the polar region of apposed lipid matrix (1 LPC:3MG:3 FA or variation thereof) monolayers is the region that binds bothwater, perhaps as water clusters (Gregory et al., Science 275:814-817,1997), and metal ions which can affect an intramolecular stabilizationof the lipid matrix.

Viscosity of LYM-X-SORB™ (LXS™) compositions were measured with aBrookfield viscometer (Model HB, Spindle CP52, Brookfield EngineeringLaboratories, Middleboro, Mass.). The LXS™ had a molar ratio ofLPC/MG/FA of (1:4:2) and prepared according to U.S. Pat. No. 5,716,814(Yesair). Water and LXS™ were added to screw-cap vials, heated at 50° C.for a half hour with shaking. The samples were allowed to cool to roomtemperature.

The viscosity data is listed in Table 2 and plotted against mole ratioof water/LXS in FIG. 8.

TABLE 2 Viscosity of LYM-X-SORB ™ with Variable Water Content MOLELYM-X-SORB WATER ADDED RATIO VISCOSITY SAMPLE # mmol LXS mmol H₂O   mmol H₂O H₂O/LXS (cP) 6-1 4.02 2.07 NONE 0.00 0.51 1780 6-2 4.00 2.070.39 2.18 1.06 1633 6-3 4.03 2.07 0.76 4.25 1.58 1558 6-4 4.03 2.07 1.025.78 1.95 2448 6-5 4.01 2.07 1.47 8.03 2.59 4971 6-6 4.02 2.07 2.1912.47 3.32 20847 6-7 4.02 2.07 2.77 15.90 4.47 31679 6-8 4.01 2.07 3.5220.33 5.59 29379 6-9 4.01 2.07 4.29 24.96 6.74 30714  6-10 4.01 2.075.25 30.80 8.20 31531

It is readily apparent that the addition of water affects changes in thelipid matrix organization as demonstrated by the marked increase inviscosity.

Sample 6-1 is similar to the sample described under the birefringentcharacteristics section of Example 2. The taste of sample 6-1 wasunpleasant with an aftertaste, whereas the taste of sample 6-10 wasunremarkable, e.g., bland with no aftertaste. Thus it can be assumedthat water can affect some organization within the LXS™ matrix.

X-ray Diffraction Analyses

In initial x-ray diffraction studies, a neat synthetic lipid matrixcontaining only the oleoyl (18:1) species of lysophosphatidylcholine(LPC; Avanti Polar Lipids, Alabaster, Ala.), monoglyceride (MG), andfatty acid (FA; Nu-Chek Prep, Inc., Elysian, Minn.) with a molar rationof 1:4:2 was evaluated. Based upon the viscosity changes of the lipidmatrix upon the addition of water (see FIG. 8), this lipid matrixcontaining ˜0, 1, 3, or 8 moles of water per mole of matrix monomer wasevaluated. In the neat (0.2% water) synthetic lipid matrix (18:1species), the x-ray diffraction pattern [obtained by Prof. ThomasMcIntosh (Duke University)] displayed 6 reflections that index as thefirst 6 orders of a lamellar (bilayer) spacing of 5.0 nm (50 Å, d_(lam),see FIG. 9A). Also present were several sharp wide-angle reflections at0.46, 0.43, and 0.40 nm corresponding to the spacing of the hydrocarbonchains. Sharp wide-angle reflections are characteristic of solid (gel)phase bilayers (see FIG. 9A). In addition, there was also present a 3.35nm (33.5 Å, d_(hex) see FIG. 9C) low-angle reflection and a 0.45 nm wideangle reflection which correspond to fluid hexagonal phase.

The fully hydrated sample (8 moles water/mole lipid matrix) was analyzedby x-ray diffraction as well as by light microscopy withcrossed-polarizers. The hydrated sample was highly birefringent withlarge regions of striations or brush patterns. Because of this intensebirefringence this phase can not be cubic phase as might have beenpredicted by the high viscosity of the hydrated lipid matrix. Thestriations are typical of hexagonal phases. In over-exposed x-raypatterns the sole wide-angle reflection is a broad band at 0.45 nm,consistent with melted hydrocarbon chains. There are no indications ofsharp wide-angle reflections. However, a very weak low-angle reflectionwas detected with long exposures at 1.94 nm in addition to the extremelystrong reflection at 3.35 nm. The spacings of these two low anglereflections have the ratio of the square root of three, expected for thefirst two orders of a hexagonal phase. Moreover, when using a fine,focused x-ray beam the 3.35 nm reflections are recorded on a hexagonallattice. Thus, the fully hydrated synthetic lipid matrix is completelyhexagonal (See FIG. 9B, 9C for schematic representations).

The samples containing 1 and 3 moles water per mole lipid matrix hadsimilar low-angle and sharp wide-angle reflections as noted for thesolid (gel) phase bilayer but much weaker than observed in the neatlipid matrix. Thus, the samples with 1 and 3 moles water per mole oflipid matrix contained both solid (gel) phase bilayer and hexagonalphase. The bilayer phase was more prominent in the sample containing 1mole water per mole lipid matrix whereas the hexagonal phase was moreapparent at 3 moles water per mole lipid matrix.

Based on the analysis described above, the addition of 8 moles of waterto the lamellar, bilayer structure of neat lipid matrix with heataffects a complete structural rearrangement to the hexagonal phase.X-ray data was collected at room temperature indicating the hexagonalphase was also stable at the lower temperature.

Thus the hexagonal phase was present in the “neat” (0.2% water) lipidmatrix, increasing with additional moisture content (1 and 3 moles ofwater per mole of lipid matrix) until only the hexagonal phase wasapparent at 8 moles per mole lipid matrix. Since varying amounts of ahexagonal phase were present in all tested lipid matrix compositions,including compositions of very low water content, it is possible thatthe water was not evenly distributed in the lipid matrix but wasdistributed as 8 moles per mole lipid matrix in the hexagonal phase and0 moles per mole lipid matrix in the lamellar phase. Alterations in theequilibrium of the water molecules in the lipid matrix between lamellarand hexagonal phases required an elevated temperature and time.

The hexagonal phase can potentially organize as a normal hexagonal (FIG.9B) or as an inverse hexagonal (FIG. 9C). The inverse hexagonal (FIG.9C) is probably more consistent with the hexagonal lattice spacing(d_(hex)) of 33.5 Å observed from the x-ray patterns. Also, the inversehexagonal phase is consistent with the geometrical arrangement of thelipid constituents having a small head group relative to the hydrocarbonchains. This structural rearrangement from lamellar to hexagonal isshown in FIG. 10. The addition of water with heat to the lamellar lipidmatrix may provide the necessary energy to affect thislamellar-hexagonal transition.

The methods used by Rand & Fuller (Biophys J., 66:2127-2138, 1994) tocharacterize the transition of dioleoylphosphatidylethanolamine (DOPE)from a lamellar phase to a hexagonal phase are employed to provideinsight into the organized lamellar and hexagonal structures of thelipid matrix. Understanding this phase transition may be useful incharacterizing the taste of the matrix with respect to lipidstructure(s) as well as in using the different organized structures fordrug delivery.

Structural Integrity of the Lipid Matrix

To correlate the physical properties of the lipid matrix with itscomposition, the following experiments are performed. First, thestructural integrity of protonated and ionized lipid matrix formulationscontaining varying molar concentrations of water, specifically 0.5, 1.0,2.0 and 3.0 moles of water per mole of the monomeric lipid matrix (3moles of water/mole of matrix represents 2.25% water) are determined.Second, the structural integrity of protonated and ionized lipid matrixformulations containing multivalent ions, specifically 0.5; 1.0, and 2.0moles of ion per mole of the monomeric lipid matrix containing water(possible water content of 0.5, 1.0, 2.0, and/or 3.0 moles of water permole of monomeric lipid matrix) are determined. The multivalent ionsthat are tested include calcium, magnesium, iron, and zinc (Nutr. Rev.,42: 220-222, 1984; Koo et al., Am. J. Clin. Nutr. 42:671-680, 1985; Kooand Turk, J. Nutr. 107:909-919, 1977; J. Nutr. 107:896-908, 1977).

The structural integrity of the lipid matrix is evaluated using thefollowing analyses:

i. Differential scanning calorimetry (DSC)

ii. X-ray diffraction

iii. ³¹P-NMR

iv. Polarizing light microscopy and viscosity

i. Differential scanning calorimetry (DSC): Based upon preliminaryfindings, the addition of water to the lipid matrix decreased themelting temperature and the addition of calcium ions further decreasedthe melting temperature. The effect of water and calcium on the matrixmelting point is difficult to rationalize if a single phase wereinvolved; but might be rationalized if water and calcium ion resulted information of a different phase (e.g., lamellar-to-hexagonal,lamellar-to-cubic). Thus the characterization of phase transitiontemperature from ordered to disordered phases of the lipid matrix in thepresence of varying water and/or metal ion content provides informationfor selecting the appropriate compositions and temperature ranges forthe x-ray diffraction and P NMR studies.ii. X-ray diffraction: We have proposed that the organized lipid matrixis lamellar. The electron dense regions of the polar headgroups of thebilayer should be separated by about 30-35 Å, i.e., the length of thenon-polar acyl hydrocarbon bilayer. Another possibility based upon theorganized structure of LPC (Saunders, Biochim. Biophys. Acta 125:70-74,1966; Hauser, J. Coll. Interf. Sci. 55:85-93, 1976) is that thehydrocarbons interdigitate to form a more condensed organization (Huiand Huang, Biochemistry 25:1330-1335, 1986).

In either organization the electron dense regions of apposed polarheadgroups would be separated by about 20-25 Å. There is also thepossibility that both types of structures exist in the more complexorganization of a cubic phase. Varying the water and ion content of thelipid matrix will probably modulate the distances between apposed polarregions. The presence of metal ions may also affect a phase change aswell.

iii. ³¹P-NMR: The resonance characteristics of phosphorus in the LPCheadgroup are influenced by the proximity of charged compounds withinthe bilayer (e.g., chorine, fatty acids) and the proximity of the groupswithin the apposing bilayer. The molecular distance between the bilayerscan be influenced by the presence of water, the protonation of the polarheadgroups, and the salt formation of the phosphate group with metalions.iv. Polarizing light microscopy and viscosity: Both of these analysesprovide data on the structural integrity of the lipid matrix (Robinsonand Saunders, J. Pharm. Pharmacol. 11:304-313, 1959; Rosevear, J. Amer.Oil Chem. Soc. 31:628-639, 1954) and also provide utilitarianmeasurements for recognizing those structural features having desirabletaste profiles. Both of these analyses represent test completion timesof less than 30 minutes and thus would be useful in defining theendpoint of the reaction process for producing the optimum lipid matrix.

The foregoing test methods are used to identify those parameters whichpredict the most stable structural integrity of the lipid matrix andprovide insight into the organization (intramolecular stabilization) ofsuch a lipid matrix.

Example 3 LYM-X-SORB™ Uses Palatable Taste Characteristics

The addition of calcium ions and/or water in defined molar ratiosrelative to the lipid matrix are factors that contribute to a morepalatable lipid matrix formulation for use as a nutritional supplement,e.g. LYM-X-SORB™ (BioMolecular Products, Inc., Byfield, Mass.), for usein cystic fibrosis (CF). It is known that polymorphic changes in PC andMG depend on the thermal history, the rate of cooling, the temperatureof equilibrium and other factors (Small, The Physical Chemistry ofLipids from Alkanes to Phospholipids, Handbook of Lipid Research 4,Plenum Press, New York, N.Y., 1986, pp. 386-392, 475-517). Thus, thephysical chemistry of the product needs to be better defined in order tocontrol the final palatability of the organized lipid matrix. Using theanalytical methods described above (x-ray diffraction, differentialscanning calorimetry and phosphorus NMR data analysis), the phasebehavior and organization (intramolecular stabilization) of the lipidmatrix in the presence of water and metal ions is assessed and comparedto palatability of the lipid matrix formulations to determine thecomposition and physical properties of the most palatable lipid matrixformulations.

For example, the taste profiles that were noted in Example 2 for Samples6-1 and 6-10, can be correlated with the X-ray diffraction analysis(Example 2) for comparable LXS™ containing similar water content. Sample6-1, low water content and undesirable taste, has a lamellarorganization. In contrast, sample 6-10 having 8 moles of water per moleLXS and good taste, is expected to have an inverse hexagonalorganization. It is reasonable to conclude that the polar head groupsaffect the undesirable taste profile and that burying the polar headgroups within the hydrophobic regions minimizes the undesirable taste ofthe head groups.

The lipid matrix (LYM-X-SORB™) can be formulated with protein,starch/carbohydrate, and flavors as wafer bars, candy bars, spray-driedproducts and ice cream. These non-lipid components, however, can alsoreduce the undesirable taste of the lipid matrix. The formulation of anintramolecularly stabilized lipid matrix nutritional supplement foranalysis of physical properties and taste characteristics is initially adried product containing 33% by weight of the lipid matrix, 18% protein,and 49% carbohydrate, starch and flavors.

Using the Littleford/Day 5 liter reactor, both a basic LXS™ matrix andan acidic LXS™ matrix were premixed with 8 moles of water per mole ofLXS™ at an elevated temperature (50-60° C.). To each, a premix ofprotein (egg white, 25% by weight), sugar (fructose, 25% by weight), andstarch (Capsul®, modified corn starch, National Starch and Chemical,Indianapolis, Ind.; 25% by weight) was added, mixed for 15-30 minutes toyield a powdered LXS™ formulation. The taste of the formulation using anaqueous basic LXS™ was bitter, whereas, the aqueous acidic LXS™ wasbland. It is presumed that aqueous basic LXS™ had formed a normalhexagonal sructure (FIG. 9B) and that aqueous acidic LXS™ had formed aninverse hexagonal structure (FIG. 9C). The inverse hexagonal structureminimizes the surface area of the polar head groups and therefore, mightminimize the undesirable taste of these groups.

The ability to use a reactor vessel, such as the Littleford/Day 5 literreactor, to prepare a powdered LXS™ formulation including the LXS™ lipidmatrix, starch, sugars and protein, reduces the loss associated withtransferring the LXS™ lipid matrix from a reaction vessel to a secondvessel for mixing with the other components of the LXS™ formulation(protein, sugar, starch, etc.). Accordingly, the cost of preparation ofthe LXS™ formulation is reduced.

The desirable palatable taste characteristics of the lipid matrix arerelated to measurable physical and structural parameters of theintramolecularly stabilized matrix using the foregoing analytical testmethods.

Standard methodology is used to quantitate the mole ratio of LPC/MG/FAof the lipid matrix, the fatty acid profile of the components and thepolyunsaturated fatty acid (PUFA) content (e.g., linoleic/linolenicration of 5:1 and >50% of fatty acid content), moisture and metal ionconcentrations, etc. The samples used for taste testing are analyzed forheavy metal and microbial limits. In addition, polarizing lightmicroscopy and viscometry methodologies are used to provide measurementsfor recognizing those structural features having desirable tasteprofiles.

Drug Delivery Formulations

In previous drug/LXS™ formulation studies (see U.S. Pat. Nos. 4,874,795;5,891,466; and 5,707,873), the LXS™ matrix was prepared using highlypurified components (LPC, MG and FA in a 1:3:3 mole ratio) containingminimal moisture content (approximately 0.5% by weight). Based uponX-ray diffraction results (see Example 2, above) the organization of theprevious LXS™ matrix is likely lamellar and any drug would be includedin the hydrophobic region of LXS™ matrix monomeric structure. Based onthe surprising results described herein that the organized molecularstructure of the hydrated LXS™ (8 moles of water per LXS monomer) is aninverse hexagonal structure, it can be seen that different structurallydiverse drugs can be incorporated within the aqueous phase of theinverse hexagonal structure (see FIG. 9C). In addition, theincorporation of drugs within the hydrophobic region of a normalhexagonal structure (see FIG. 9B) would result in more biologicallystable drug/LXS™ formulations within the hostile environments of thegastrointestinal tract. Furthermore, the lamellar, normal hexagonal andinverse hexagonal organization of LXS™ compositions containing drug(s)would also be useful for many routes of administration, e.g., dermal,inhalation, suppository, etc.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All references, publications and patents disclosed herein areincorporated by reference in their entirety.

1. A drug delivery composition comprising a lipid matrix and apharmaceutically acceptable carrier, wherein at least part of the lipidmatrix is in a hexagonal phase or an inverse hexagonal phase.
 2. A drugdelivery composition comprising a lipid matrix and a pharmaceuticallyacceptable carrier, wherein at least part of the lipid matrix is in aphase other than a lamellar phase, hexagonal phase or inverse hexagonalphase.
 3. The drug delivery composition of claim 1, wherein the lipidmatrix comprises from about 0 to about 8 moles of water per mole oflipid.
 4. The drug delivery composition of claim 3, wherein the lipidmatrix comprises at least about 1 moles of water per mole of lipid. 5.The drug delivery composition of claim 3, wherein the lipid matrixcomprises at least about 3 moles of water per mole of lipid.
 6. The drugdelivery composition of claim 3, wherein the lipid matrix comprises atleast about 8 moles of water per mole of lipid.
 7. The drug deliverycomposition of claim 1, wherein the lipid matrix compriseslysophosphoglyceride, monoglyceride and fatty acid, wherein the molarratio of lysophosphoglyceride:the sum of monoglyceride and fatty acid inthe lipid matrix is between 1:3 and 1:12.
 8. The drug deliverycomposition of claim 7 wherein the molar ratio oflysophosphoglyceride:the sum of monoglyceride and fatty acid in thelipid matrix is between 1:5 and 1:6.
 9. The drug delivery composition ofclaim 8 wherein the lipid matrix has alysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2and 1:2:4.
 10. The drug delivery composition of claim 9 wherein thelipid matrix has a lysophosphoglyceride:monoglyceride:fatty acid molarratio selected from the group consisting of 1:4:2, 1:3:3 and 1:3:2. 11.The drug delivery composition of claim 7, wherein thelysophosphoglyceride is lysophosphatidylcholine.
 12. The drug deliverycomposition of claim 1, further comprising one or more water soluble orwater insoluble pharmaceutical compounds.
 13. The drug deliverycomposition of claim 2, wherein the lipid matrix comprises from about 0to about 8 moles of water per mole of lipid.
 14. The drug deliverycomposition of claim 13, wherein the lipid matrix comprises at leastabout 1 moles of water per mole of lipid.
 15. The drug deliverycomposition of claim 13, wherein the lipid matrix comprises at leastabout 3 moles of water per mole of lipid.
 16. The drug deliverycomposition of claim 13, wherein the lipid matrix comprises at leastabout 8 moles of water per mole of lipid.
 17. The drug deliverycomposition of claim 2, wherein the lipid matrix compriseslysophosphoglyceride, monoglyceride and fatty acid, wherein the molarratio of lysophosphoglyceride:the sum of monoglyceride and fatty acid inthe lipid matrix is between 1:3 and 1:12.
 18. The drug deliverycomposition of claim 17 wherein the molar ratio oflysophosphoglyceride:the sum of monoglyceride and fatty acid in thelipid matrix is between 1:5 and 1:6.
 19. The drug delivery compositionof claim 18 wherein the lipid matrix has alysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2and 1:2:4.
 20. The drug delivery composition of claim 19 wherein thelipid matrix has a lysophosphoglyceride:monoglyceride:fatty acid molarratio selected from the group consisting of 1:4:2, 1:3:3 and 1:3:2. 21.The drug delivery composition of claim 17, wherein thelysophosphoglyceride is lysophosphatidylcholine.
 22. The drug deliverycomposition of claim 2, further comprising one or more water soluble orwater insoluble pharmaceutical compounds.
 23. A drug deliverycomposition comprising a lipid matrix and a pharmaceutically acceptablecarrier, wherein at least part of the lipid matrix is in a lamellarphase.
 24. The drug delivery composition of claim 23, wherein the lipidmatrix comprises lysophosphoglyceride, monoglyceride and fatty acid,wherein the molar ratio of lysophosphoglyceride:the sum of monoglycerideand fatty acid in the lipid matrix is between 1:3 and 1:12.
 25. The drugdelivery composition of claim 24 wherein the molar ratio oflysophosphoglyceride:the sum of monoglyceride and fatty acid in thelipid matrix is between 1:5 and 1:6.
 26. The drug delivery compositionof claim 25 wherein the lipid matrix has alysophosphoglyceride:monoglyceride:fatty acid molar ratio between 1:4:2and 1:2:4.
 27. The drug delivery composition of claim 26 wherein thelipid matrix has a lysophosphoglyceride:monoglyceride:fatty acid molarratio selected from the group consisting of 1:4:2, 1:3:3 and 1:3:2. 28.The drug delivery composition of claim 24, wherein thelysophosphoglyceride is lysophosphatidylcholine.
 29. The drug deliverycomposition of claims 23, further comprising one or more water solubleor water insoluble pharmaceutical compounds.
 30. A nutritionalsupplement for the treatment of cystic fibrosis comprising an effectiveamount of the drug delivery composition of any of claims 1, 2 or
 23. 31.A nutritional supplement comprising the drug delivery composition of anyof claims 1, 2 or 23.